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ELISA Antibody Pair Protein Serine/Threonine Kinase Activity

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Phospho-Akt (Thr308) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252. Capture and Detection Antibodies (100X stocks) and HRP-Conjugated Secondary Antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Akt Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added, followed by Phospho-Akt (Thr308) Mouse Detection Antibody and HRP-conjugated Anti-Mouse IgG. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-Akt (Thr308) protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse

Background: Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160. Capture and Detection antibodies (100X stocks) and HRP-Conjugated Secondary Antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Phospho-Akt (Ser473) Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by Akt1 Mouse Detection Antibody and HRP-conjugated Anti-Mouse IgG. HRP substrate (TMB) is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-Akt1 (Ser473) protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse, Rat

Background: Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Total SAPK/JNK Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Total SAPK/JNK Sandwich ELISA Kit #7330. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The SAPK/JNK Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by SAPK/JNK Detection Antibody and Anti-Rabbit IgG, HRP-conjugated antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of total SAPK/JNK protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human

Background: The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK is potently and preferentially activated by a variety of environmental stresses including UV and gamma radiation, ceramides, inflammatory cytokines, and in some instances, growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-MEKK4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4/7. Upon activation, MKKs phosphorylate and activate the SAPK/JNK kinase (2). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (3). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (3). Alternatively, MKK4/7 can be activated in a GTPase-independent mechanism via stimulation of a germinal center kinase (GCK) family member (4). There are three SAPK/JNK genes each of which undergoes alternative splicing, resulting in numerous isoforms (3). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (3,5).

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Total Akt1 Sandwich ELISA Antibody Pair is being offered as an economical alternative to our PathScan® Total Akt1 Sandwich ELISA Kit #7170. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Akt Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by Akt1 Mouse Detection Antibody and HRP-conjugated Anti-Mouse IgG. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Total Akt1 protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse

Background: Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit #7325. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Phospho-SAPK/JNK (Thr183/Tyr185) Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by SAPK/JNK Detection Antibody and Anti-mouse IgG, HRP-conjugated Antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-SAPK/JNK (Thr183/Tyr185) protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse

Background: The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK is potently and preferentially activated by a variety of environmental stresses including UV and gamma radiation, ceramides, inflammatory cytokines, and in some instances, growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-MEKK4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4/7. Upon activation, MKKs phosphorylate and activate the SAPK/JNK kinase (2). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (3). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (3). Alternatively, MKK4/7 can be activated in a GTPase-independent mechanism via stimulation of a germinal center kinase (GCK) family member (4). There are three SAPK/JNK genes each of which undergoes alternative splicing, resulting in numerous isoforms (3). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (3,5).

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-AMPKα-(Thr172) Sandwich ELISA Kit #7959. Capture and Detection antibodies (100X stocks) and Anti-Mouse IgG, HRP-linked Antibody (1000X stock) are supplied. Sufficient reagents are provided for 4 x 96 well ELISAs. The AMPKα Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a Phospho-AMPKα (Thr172) Mouse Detection Antibody and Anti-Mouse IgG, HRP-linked Antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-AMPKα (Thr172) protein.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse

Background: AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation (3-5). AMPKα is also phosphorylated at Thr258 and Ser485 (for α1; Ser491 for α2). The upstream kinase and the biological significance of these phosphorylation events have yet to be elucidated (6). The β1 subunit is post-translationally modified by myristoylation and multi-site phosphorylation including Ser24/25, Ser96, Ser101, Ser108, and Ser182 (6,7). Phosphorylation at Ser108 of the β1 subunit seems to be required for the activation of AMPK enzyme, while phosphorylation at Ser24/25 and Ser182 affects AMPK localization (7). Several mutations in AMPKγ subunits have been identified, most of which are located in the putative AMP/ATP binding sites (CBS or Bateman domains). Mutations at these sites lead to reduction of AMPK activity and cause glycogen accumulation in heart or skeletal muscle (1,2). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (1).

$469
Reagents for 4 x 96 well plates
1 Kit
CST's PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177. Capture and detection antibodies (100X stocks) and an HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Phospho-p44/42 MAPK Rabbit Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysate is added followed by a p44/42 MAPK Mouse Detection Antibody and an HRP-conjugated Anti-Mouse IgG Antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of p44/42 MAPK phosphorylated at Thr202/Tyr204.Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse

Background: Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.

$469
Reagents for 4 x 96 well plates
1 Kit
Cell Signaling Technology's PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063. Capture and detection antibodies (100X stocks) and an HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The p70 S6 kinase rabbit capture antibody is coated onto a 96 well microplate overnight in PBS. After blocking, cell lysates are added followed by a phospho-p70 S6 kinase (Thr389) mouse detection antibody and anti-mouse IgG, HRP-linked antibody. HRP substrate (TMB) is then added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-p70 S6 kinase (Thr389).Antibodies in kit are custom formulations specific to kit.
REACTIVITY
Human, Mouse

Background: p70 S6 kinase is a mitogen activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression (1,2). p70 S6 kinase phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). A second isoform, p85 S6 kinase, is derived from the same gene and is identical to p70 S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal (1). Both isoforms lie on a mitogen activated signaling pathway downstream of phosphoinositide-3 kinase (PI-3K) and the target of rapamycin, FRAP/mTOR, a pathway distinct from the Ras/MAP kinase cascade (1). The activity of p70 S6 kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains (1). Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function (1). Phosphorylation of Thr389, however, most closely correlates with p70 kinase activity in vivo (3). Prior phosphorylation of Thr389 is required for the action of phosphoinositide 3-dependent protein kinase 1 (PDK1) on Thr229 (4,5). Phosphorylation of this site is stimulated by growth factors such as insulin, EGF and FGF, as well as by serum and some G-protein-coupled receptor ligands, and is blocked by wortmannin, LY294002 (PI-3K inhibitor) and rapamycin (FRAP/mTOR inhibitor) (1,6,7). Ser411, Thr421 and Ser424 lie within a Ser-Pro-rich region located in the pseudosubstrate region (1). Phosphorylation at these sites is thought to activate p70 S6 kinase via relief of pseudosubstrate suppression (1,2). Another LY294002 and rapamycin sensitive phosphorylation site, Ser371, is an in vitro substrate for mTOR and correlates well with the activity of a partially rapamycin resistant mutant p70 S6 kinase (8).